The present invention relates to the enzyme catalyzed replication of large biological molecules and in particular relates to microwave assisted PCR amplification of DNA.
As well understood by those of ordinary skill in this art, and indeed to some extent by the layperson, the DNA molecule carries the genetic code that determines the physical features and functions of biological organisms, including those of human beings. The DNA molecule can be described as a double helix in which phosphate groups and sugars form two respective chains (“backbones”) while amino groups (typically referred to as “bases”) attached to the chains form hydrogen bonds with one another that connect the two chains (“strands”) together. Because the bases form hydrogen bonds with geometric specificity, the bases always connect to one another in specific pairs. As a result, when the strands separate (or are separated), each provides a pattern of successive bases that form a template for reconstructing the entire DNA molecule from either, or both, of the separated strands. This process is, of course, carried out naturally in living organisms and is referred to as “replication.”
In many circumstances, DNA is available in extremely small quantities. When such quantities are too small for identification or analysis, the DNA can be amplified by a laboratory practice that is patterned after natural DNA replication and that is referred to as the “polymerase chain reaction” and typically abbreviated as “PCR.” PCR is generally carried out in three steps with one set of such steps being referred to as a “cycle.” For comparison purposes, PCR results are usually best understood in terms of the results that can be obtained after at least about 30 PCR cycles.
U.S. Pat. Nos. 4,683,202 and 4,683,195 are often referred to as seminal references in describing PCR technology. U.S. Pat. No. 4,683,202 has been cited in over 1400 later U.S. Patents and U.S. Pat. No. 4,683,195 has been cited in over 1300 later U.S. Patents. Accordingly, the basic, and to a great extent the sophisticated, aspects of PCR amplification are well understood in this art and will not be described in detail other than is necessary to describe the invention herein.
The capacity to amplify DNA creates the related capacity to evaluate DNA for a number of diagnostic purposes. PCR can produce DNA samples of sufficient size to detect the presence or absence of particular items, typically a virus, a bacterium, or even a particular sequence of genetic material. PCR technology thus provides the capacity to detect infectious diseases and genetic characteristics including genetic variations and mutations. PCR is accordingly the basis for a number of clinical diagnostic tests for infectious diseases. Because PCR is extremely sensitive, in some cases it can also quantify (in addition to identifying) the amount of a particular virus in a person's blood, thus providing the capacity to monitor either the progression of a disease or its response to treatment. For the same reasons, PCR is extremely valuable in blood screening (i.e., donated blood) to identify or preclude the presence of infectious agents. From a genetic testing standpoint, PCR can identify a genetic predisposition toward particular diseases or conditions and in some cases can, or is expected to, identify or predict how a particular person will respond to a specific pharmaceutical (or other medical) treatment. See, Roche Molecular Diagnostics, Applications of PCR [Online], www.Roche-Diagnostics.com/ba_rmd/pcr_applications.html, March 2006.
DNA amplification also provides a relatively straightforward method of purifying a particular DNA segment that is otherwise present in a bulk material. If the amplification is carried out to a sufficient extent, the desired DNA product becomes a proportionally overwhelming component of the mixture, thus effectively reducing other contaminants to trivial amounts. See, Cantor, Genomics, John Wiley and Sons, Inc., 1999, at page 98.
As yet another advantage, PCR amplification of DNA can be used to create libraries of DNA that in turn can be used in combinatorial chemistry techniques for a variety of analytical, diagnostic or synthesis purposes.
Each PCR cycle includes the steps of (i) denaturation, (ii) annealing, and (iii) extension. Prior to denaturation, the DNA which is to be amplified is mixed in combination with primer molecules and enzymes.
“Denature” refers to the step of separating the DNA double helix into two individual strands with a goal of at least 99 percent completion. In typical PCR techniques, the denaturation step is carried out by heating the DNA to a temperature of between about 90 and 105° C. for a period of between about one and ten minutes, at which temperature the double strand opens to form single stranded DNA. This temperature also tends to stop reactions from the previous cycle.
“Annealing” refers to the step of adding specific primers to the separated DNA strands. Primers are required because the DNA enzymes cannot start DNA chains from scratch. Instead, the primer is required to determine the location along a particular DNA template at which the synthesis of the complementary strand will begin. Thus, specific desired DNA regions can be selectively amplified by using appropriate primers. Primers are short, synthetic sequences of single-stranded DNA, typically consisting of 20-30 bases, with a labeled end structure to aid in identification. They are generally specific for the target region of the DNA of the organism. In most PCR amplifications, two primers are used, one for each of the complementary single DNA strands that was produced during denaturation. The art is replete with examples of specific primers that have been developed to amplify specific types or portions of DNA.
The annealing step is carried out by lowering the temperature to between about 50 and 60° C. at which point the primers attach themselves in an appropriate fashion and amount to the individual DNA strands that were previously separated.
Once the annealing step has been carried out to an extent that binds the primers to the DNA strands, the temperature is again raised, typically to greater than 70° C. and an enzyme is used to help replicate the DNA strands. The enzyme synthesizes new double-stranded DNA molecules by facilitating the joining of the complementary nucleotides (i.e., the sugar joined to a base and to a phosphate group) in solution.
As a result, at the end of the first PCR cycle, two new DNA strands are present, each of which is identical to the original target DNA strand that was denatured and primed.
As noted above, a typical DNA amplification requires about 30 PCR cycles. From a time standpoint, the denaturation step typically takes about two minutes, the cooling step that anneals the primers onto the separate strands likewise takes about two minutes, and the extension step again takes about two minutes. Thus each PCR cycle takes on the order of about six minutes. In turn, a 30 cycle amplification will take between about two and three hours of total time.
As in any other process of scientific or commercial importance, reducing the time required for any one or more of the steps will likewise reduced the time required to carry out one cycle and thus reduce the time required for the total amplification. Furthermore, because each step in the PCR cycle is a thermal step, the time required will typically increase based upon increased amounts of material.
Accordingly, the time required to carry out any one or more of the PCR steps can create a corresponding disadvantage in DNA amplification and replication.
As another factor, because the typical PCR cycle (e.g. using Taq polymerase; Cantor, supra) includes moving the temperature of the compositions upwardly (denaturation at 92-96° C. for 30-60 seconds) and then downwardly (annealing at 55-60° C. for about 30 seconds) and then upwardly again (extension at about 72° C. for about 60 seconds), fluctuations in the temperatures can cause undesired modifications that are rapidly magnified. As generally well understood in this art, a typical PCR instrument uses a plurality of tube holders or microtitre plate holders that are temperature controlled by heating elements and cooling baths, or by thermoelectric heating and cooling, or by forced convection heating and cooling, or by switching between and among water baths. See Cantor, supra at page 103.
In short, temperature swings during PCR amplification can create or increase undesired modifications.
As yet another factor, because the denaturing step requires the relatively higher temperature, the other compounds present in the PCR sample, and most typically the extension enzymes, must remain stable at these elevated denaturation temperatures. Accordingly, these temperature requirements effectively limit the selection of available enzymes for PCR.
In particular, the Taq polymerase is used because it can generally withstand the DNA denaturation temperatures. In conventional PCR, using an enzyme that cannot withstand the denaturation temperatures requires that fresh enzyme he added after every cycle, thus increasing the manipulative complexity of the process and the time required.
Although using high temperature enzymes such as Taq eliminates the need to add fresh enzyme after every cycle, such high temperature enzymes, including Taq, raise different problems. For example, the Taq enzyme lacks a 3′ to 5′ exonuclease activity. Such activity is informally referred to as a “proofreading” capability in which the enzyme is able to identify misplaced bases and replace them with correct bases in the desired positions (in a manner consistent with natural organisms). If the PCR cycle misidentifies or misplaces bases, it will, of course, replicate something other than the target sequence. Because of the nature of amplification, such errors will be significantly magnified over the course of many cycles.
As a second problem, standard PCR is typically limited to target sequences of between about 2000 and 3000 base pairs. Conducting PCR on larger targets (e.g., up to 50,000 base pairs) tends to require slower heating cycles and special enzymes that may not be amenable (or as amenable) to conventional PCR cycles.
Accordingly, a lack of fidelity and size limitations in the target sequences remain as disadvantages in conventional PCR.